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High-throughput identification of proteins with AMPylation using self-assembled human protein (NAPPA) microarrays

Nature Protocols volume 10, pages 756–767 (2015)Cite this article

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Abstract

AMPylation (adenylylation) has been recognized as an important post-translational modification that is used by pathogens to regulate host cellular proteins and their associated signaling pathways. AMPylation has potential functions in various cellular processes, and it is widely conserved across both prokaryotes and eukaryotes. However, despite the identification of many AMPylators, relatively few candidate substrates of AMPylation are known. This is changing with the recent development of a robust and reliable method for identifying new substrates using protein microarrays, which can markedly expand the list of potential substrates. Here we describe procedures for detecting AMPylated and auto-AMPylated proteins in a sensitive, high-throughput and nonradioactive manner. The approach uses high-density protein microarrays fabricated using nucleic acid programmable protein array (NAPPA) technology, which enables the highly successful display of fresh recombinant human proteins in situ. The modification of target proteins is determined via copper-catalyzed azide-alkyne cycloaddition (CuAAC). The assay can be accomplished within 11 h.

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Figure 1: Distribution of Fic/DOC protein family sequences across a variety of 3,000+ species.
Figure 2: Outline of the NAPPA protocol for the detection of AMPylation substrates.
Figure 3: Quality control of self-assembled human protein NAPPA microarrays.
Figure 4: Representative results from the AMPylation screening with self-assembled human NAPPA microarrays.
Figure 5: The main steps to prepare for the in vitro transcription and translation of plasmid DNAs that are printed on NAPPA arrays.

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Acknowledgements

We thank the Early Detection Research Network (5U01CA117374). We thank K. Orth's laboratory (Department of Molecular Biology, University of Texas Southwestern Medical Center) and H. Hang's laboratory (The Laboratory of Chemical Biology and Microbial Pathogenesis, The Rockefeller University) for providing the purified AMPylator proteins and click reagents, respectively. We thank B. Petritis and K. Barker for critical reading of the manuscript.

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Authors and Affiliations

  1. The Virginia G. Piper Center for Personalized Diagnostics, Biodesign Institute, Arizona State University, Tempe, Arizona, USA

    Xiaobo Yu & Joshua LaBaer

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  1. Xiaobo Yu
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X.Y. designed and performed the experiments, and wrote the manuscript; J.L. designed the study and wrote the manuscript.

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Correspondence to Joshua LaBaer.

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Yu, X., LaBaer, J. High-throughput identification of proteins with AMPylation using self-assembled human protein (NAPPA) microarrays. Nat Protoc 10, 756–767 (2015). https://doi.org/10.1038/nprot.2015.044

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